Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents that is historically divided into two subgroups based upon molecular and histological features. One subgroup is the alveolar PAX3/7-FOXO1 fusion containing tumors and the other embryonal, fusion-negative tumors. Overall, treatment regimens for RMS include surgery, radiation and cytotoxic chemotherapy, which have improved overall survival rates to approximately 65-70%. However, despite intensification of therapeutic regimens, long- term outcomes for patients with metastatic or relapsed rhabdomyosarcoma remain extremely poor, with overall survival rates between 20-30%, thus alternative targeted therapies, or combination therapy is essential for enhancing outcomes. Recent efforts to understand the genomic landscape of RMS have identified key mutations in potent drivers of oncogenesis. Whole exome sequencing of RMS xenografts identified mutations in the RAS/NF1 pathways in approximately 45% of cases, predominantly within the fusion-negative population. Of the patients with RAS mutations, 75% are considered high-risk patients. The p21-activated kinases (PAK) family of proteins intersects the oncogenic signaling pathways activated by RAS, and downstream PAK signaling activates key cell processes involved in driving metastatic phenotypes and resistance to therapeutics, thus making them attractive targets for advanced cancers. Our data suggests that PAKs are activated in RMS and small molecule inhibition of PAKs have significant anti-tumor activity. We hypothesize that combination therapy inhibiting PAK activity with a probe drug that targets a different network or cellular process will create a synergistic therapeutic pair that is highly cytotoxic to RMS cells. To address this hypothesis and combat the significant therapeutic deficiencies we propose two innovative, translational and exploratory aims. Aim1 will apply an innovative and comprehensive PAK inhibition anchor-probe based synthetic lethality screen in order identify candidate synergistic therapies for the treatment of high-risk RMS. Subsequently, we will assess the in vitro and ex vivo efficacy of these single agent and combination therapies for targeting metastatic phenotypes and ability to inhibit metastatic colonization and macroscopic development using a 3D ex vivo pulmonary metastasis assay. Aim2 will apply our extensive inventory of pre-clinical models, including (1) resources from an immunocompetent conditional genetically engineered mouse model (GEMM) with alterations of Ras and p53 that mimics the high risk disease state and (2) representative patient-derived xenografts (PDXs) to assess the efficacy of synergistic combination therapies for the treatment of high-risk RMS. Our research will provide essential pre-clinical studies investigating this family of kinases as novel, viable therapeutic targets for the treatment of high-risk RMS that can be quickly moved towards clinical trial development.